This grant describes a series of genetic and biochemical experiments designed to probe the interaction between DNA binding proteins and their recognition sites in double-stranded DNA. The cI, c2, cro, arc, and mnt repressors of bacteriophage Lambda and P22 will be studied. We will also study the inactivation of the Lambda cI repressor and the P22 c2 repressor by the E. coli RecA protein and by the P22 antirepressor protein. The long range goal of this research is to understand how proteins recognize specific sequences of DNA, and to understand how interaction between DNA binding proteins and other proteins regulates DNA binding. Lambda repressor mutants which are properly folded but which show a reduced affinity for operator DNA will initially be identified. We will purify these mutant proteins and will assay their interaction with operator DNA by methylation protection and nuclease protection techniques. The binding of these mutant repressors to non-operator DNA will also be assayed. Such experiments may allow us to identify specific points of contact between the protein and DNA by differences in protection patterns, and should allow an estimate of the energetic loss resulting from perturbation of specific protein-DNA contacts. Similar experiments will also be performed with Lambda cro protein. Since the three-dimensional structures of the Lambda cro protein and the N-terminal, operator binding domain of Lambda repressor are known, our mutant studies should be interpretable in detailed molecular terms. The arc and mnt repressors have yet to be obtained in pure form. We will purify these repressors and will study their interaction with operator DNA. The affects of these proteins on P22 immunity I transcription in vitro will also be determined. Mutants of Lambda repressor resistant to recA cleavage, P22 antirepressor inactivation, and inactivation by dominant negative repressor mutants will be selected and sequenced. We will use these purified mutant repressors, and proteolytic fragments of wild-type repressor to study the mechanism by which the C-terminal domain of repressor mediates RecA cleavage, antirepressor binding, oligomerization, and binding of heteroimmune repressors.